ABSTRACT
Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.
Subject(s)
Bacterial Outer Membrane Proteins/pharmacology , Cell Proliferation/drug effects , Pasteurella multocida/metabolism , Uridine Phosphorylase/isolation & purification , Uridine Phosphorylase/pharmacology , Amino Acid Sequence , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Cattle , Cell Line/drug effects , Escherichia coli/genetics , Humans , Interleukin-2/metabolism , Mice , Molecular Weight , Pasteurella multocida/genetics , Phosphorylases , Recombinant Proteins , Spleen , T-Lymphocytes/drug effects , Uridine Phosphorylase/genetics , Uridine Phosphorylase/metabolismABSTRACT
5'-Deoxy-4',5-difluorouridine (4'-F-5'-dFUrd) (10) has been synthesized on the basis of the rationale that the labilization of the glycosidic linkage caused by the 4'-fluoro substituent might allow this compound to be a better prodrug form of the anticancer drug 5-fluorouracil (FUra) than is the widely studied fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd). The rate of solvolytic hydrolysis of the glycosidic linkage of 4'-F-5'-dFUrd at pH 1 was about 500-fold faster than that of 5'-dFUrd. Since uridine phosphorylase is thought to be the enzyme that causes degradation of 5'-dFUrd in vivo to generate FUra, we compared the substrate interactions of 5'-dFUrd and 4'-F-5'-dUrd with this enzyme. The Vmax for hydrolysis of 4'-F-5'-dFUrd to FUra by uridine phosphorylase was about 5-fold greater than that of 5'-dFUrd, whereas the Km value of 4'-F-5'-dFUrd was 10-fold lower. The combination of these two factors results in 4'-F-5'-dFUrd having a 50-fold higher value of V/K than does 5'-dFUrd. Against L1210 cells in culture, the IC50 value for growth inhibition by 4'-F-5'-dFUrd was 3 X 10(-7) compared to 3 X 10(-6) for 5'-dFUrd.
Subject(s)
Antineoplastic Agents/chemical synthesis , Floxuridine/chemical synthesis , Fluorouracil/metabolism , Pentosyltransferases/pharmacology , Pharmaceutical Preparations/chemical synthesis , Prodrugs/chemical synthesis , Uridine Phosphorylase/pharmacology , Animals , Floxuridine/pharmacology , Hydrolysis , Kinetics , Mice , Mice, Inbred DBA , Prodrugs/metabolism , Prodrugs/pharmacology , Tumor Cells, Cultured/drug effectsABSTRACT
Salvage of circulating nucleosides provides an alternative to de novo synthesis of nucleotides and may modify response to antimetabolites. We have investigated treatment with uridine phosphorylase as a means of inhibiting salvage of uridine in vivo. Examination of the metabolism of intravenous [3H] uridine in mice revealed that 30-40% was salvaged by conversion to uracil nucleotides and the remainder was catabolized. In contrast, less than 0.3% of intravenous [3H]uracil was salvaged. Addition of partially purified bacterial uridine phosphorylase to plasma produced a rapid phosphorolysis of uridine. In vivo, 1.5 hr after intravenous injection of 9 units of uridine phosphorylase, plasma activity (1.3 units/ml) was 65-fold greater than that of control mice. Pretreatment with uridine phosphorylase prior to administration of [3H]uridine produced a marked (65-92%) but incomplete inhibition of salvage of uridine in all tissues examined. The dose required to produce 50% inhibition of uridine salvage at 1 hr was 2 to 2.5 units/mouse. The inhibition of nucleoside salvage by this approach may permit an evaluation of the role of nucleoside salvage in the supply of cellular nucleotides and the effects of concurrent inhibition of de novo and salvage pathways for nucleotide synthesis.
